G蛋白Rab3a cDNA的克隆与表达

利用PCR法 ,从人胎盘总cDNA中扩增得到Rab3acDNA的全编码区 .序列分析表明 ,扩增得到的Rab3acDNA有 5个核苷酸发生了变异 ,但翻译的氨基酸与发表的完全一致 .将扩增得到的Rab3acDNA克隆于原核融合表达载体pGEX 4T 1中 ,在E .coliBL2 1中经IPTG诱导表达 .为了进一步鉴定表达产物 ,对纯化后的Rab3a蛋白进行了SDS PAGE、N端氨基酸测序、质谱分子量测定及氨基酸组成分析鉴定 .结果显示 ,表达蛋白的分子量约 2 5kD ,N端氨基酸序列为MASATDSR ,氨基酸组成分析表明 ,Rab3a蛋白获得了正确表达

Cloning and Expression of G-protein Rab3a cDNA

The full coding region of Rab3a cDNA was amplified using human placenta total cDNA as template by PCR. The result from nucleotide sequencing indicated that the amplified Rab3a cDNA harbored five polymorphic nucleotides, but the deduced amino acid sequences from the nucleotide sequences were identical with the referred Rab3a protein. Afterwards, the amplified Rab3a was cloned into the prokaryotic fusion expression vector pGEX 4T 1. E.coli BL21 was transformed with this recombinant construct and induced with IPTG for expression. The purified Rab3a was further subject to SDS PAGE, N terminal amino acid residue analysis, and mass spectrography molecular weight monitoring and amino acid composition analysis. The results showed that the molecular weight of expressed protein was about 25 kD,the N\|terminal amino acid sequence was MASATDSR.The compositional analysis indicated that Rab3a protein was expressed correctly.